USPTO Patents Application 09993647

Requested Patent: EP0107214A2
Title:

PHARMACEUTICAL COMPOSITIONS CONTAINING
BENZOYL-PHENYL UREAS WITH ANTI-TUMOUR ACTIVITY. ;

Abstracted Patent: EP0107214 ;
Publication Date: 1984-05-02;

Inventor(s): MAYER RICHARD T;; JENKINS VERNON K ;

Applicant(s): DUPHAR INT RES (NL) ;

Application Number: EP1 9830201 263 19830902 ;

Priority Number(s):

US 198204 14037 19820902; US1 982041 4038 19820902 ;

IPC Classification: A61K31/17; A61K31/44 ;

Equivalents: AU 1862083 ;

ABSTRACT:

A method of and composition suitable for combating tumors in mammals is
disclosed, characterized in that a therapeutically effective quantity of at least
one 1-benzoy!-3-phenyl ureas or a metabolite thereof, which compounds are
known per se, is administered in a pharmaceutically acceptable carrier.

Europaisches Patentamt
Qjjj European Patent Office

Office europgen des brevets

© Publication number:

CM 07 214

EUROPEAN PATENT APPLICATION

Application number: 83201263.7

R) int. A 61 K 31/17, A61K 31/44

Date of filing: 02,09-83

Priority: 02,03.82 US 414037
02.09.82 US 414038

Applicant; DUPHAtt INTERNATIONAL RESEARCH B-V,
C J. van Houtenlaan 36, NL-13S1 CPWeesp (NL)

@ Date of publication of application :, 02,05,84
Bulletin 84/18

Inventor: Jenkins, Vernon IC, c/o OCTftOOIBUHEAU
ZOAN B.V, ApQifolsan 151 , NL-1077 AR Amsterdam (NL)
Inventor: Mayer, Richard T., c/o OCTROOIBUREAU
ZOAN B.V. Apoltoioart 151, NL-1077 AR Amsterdam (NL)

@ Designated Contracting States : AT BE CH DEFRGBIT
Li NL SE

@ Representative: Mute, Maarten, Drst at el,

OCTBOOIBUBEAU ZOAN B.V. ApoHoiaan 161,
NL-1 077 AR Amsterdam (NL)

@ Pharmaceutical compositions containing benzoyl-phenyl ureas wEth anti-tumour activity.

© A method of and composition suitable for combating tu-
mors in mammals is disclosed, characterized in that a thera-
peutically effectfve quantity of at least one 1~benzoy£-3-phenyi
ureas or a metabolite thereof, which compounds are known
per se, is administered in a pharmaceutical^ acceptable carri-
er.

ACTORUM AG

TITLE MGDH=|J5R)721*

see front pag-*s

Pharmaceutical compositions Having antitumor activity.

The present invention relates to novel pharmaceu-
tical compositions having antitumor activity, and to a
method of treating tumors in mammals.

United States Patent No. 3.748.356 discloses that
l-benzoyl-3-phenyl ureas have insect^cidal activity. This
activity is based on a mechanise Lhat is not fully under-
stood at this time. It has been found that the insects are
killed by these compounds -because these l~benzoyl-3~pbenyl
ureas influence, i.e., inhibit, chitin formation.

Furthermore, European Patent Application No.
0.025.363 mentions that N-benzoyl-N' -pyridyloxy phenyl
ureas have antitumor effects.

It has now been found that l-benzoyl-3-phenyl
ureas known per se with the exception of the compounds
having antitumor activity known from European patent ap-
plication No. 0.025.363, Japanese patent application No.
185.451 (publication No. 57.109.721) and Japanese patent
application No. 104765 (publication No. 57.031.664), which
have inhibitory activity on chitin formation and known
metabolites thereof, have a very interesting cytostatic or
tumor icidal effect on mammalian tumors.

More specifically, it was found that

1) i-{2,6-difluorobenzoyl)-3-(4-chlorophenyl)urea (known

as dif lubenzuron) ;

2) l-(2,6-dif luorobenzoyl)-3-i:3, 5-dichloro-4-( 3-chloro-5-

-t r i f luoromethyl-2-pyr idy loxy >pheny 1 jurea ?

3) l-{2, 6-dif luorobenzoyl)-3-t3-chloro-4-(4-chlorophenoxy)

phenyl 3 urea;

4 ) 1- ( 2 , 6-di f luorobenaoyl ) -3- ( 4-tr i f luoromethylphenyl ) urea

(known as PH 60-44) f

5 ) l- ( 2-chlorobenzoyl ) -3- ( 4-chloropheny 1 ) urea ;

6 ) 1- ( 2-chlorobenzoyl ) - 3- ( 4-tr i f luororoethoxyphenyl ) urea ;

7 ) 1- ( 2 , 6-di fluorobenzoyl ) -3- ( 4-chloro-2-hydroxyphenyl
urea;

8) l-( 2 , 6-dif luorobenzoyl) -3- {4-chloro-3-hydroxyphenyl) urea

0107214

9 ) 1- ( 2-chlorobenEoyl ) -3- { 2-hydroxy-4-tr i f luoromethoxy-
phenyl)urea

10 } 1- { 2 , 6-di f luorobenaoyl } -3- ( 4-cyanophenyl ) ur ea ;

11 ) 1- { 2 , 6-di £ luorobenzoyl ) -3- £4™ (1,1,2,3,3, 3-hexaf luoropro-
poxy ) phenyl 3 ur ea ;

12 ) 1- { 2 , 6-di f luoroben^oyl ) - 3- ( 4-hydroxyphenyl ) urea ?

13 ) 1- ( 2 , 6-di f luorobenaoyl ) - 3- ( 4-cyclohexylphenyl ) urea ?

14 ) 1™ { 2 , 6-di £ luorobertEoyl } -3- ( 4-tr ifluorome thoxyphenyl ) urea
15 ) 1- ( 2 , 6-d i fluorobensoyl } -3- ( 3 , 4-dichlorophenyl ) urea ;

16 ) 1™ ( 2-chlorobenjaoyl } -3- { 4-tri fluorome thylphenyl ) urea ;

17 ) 1- ( 2 , 6-dichlorobenzoyl ) -3- { 4-chlorophenyl ) urea ;

IS ) 1- ( 2 , 6-di chlorobenjsoyl ) -3™ ( 4-tr i fluorome thylphenyl ) urea
19 ) l-benzoyl~3- ( 4-chlor opine nyl ) urea ;

20) l-{2, 6-dif luorobenisoyl ) -3- ( 2-hy droxy- 4-tr ifluorome thy 1-
phenyl } ur ea ;

21 ) 1- ( 2-chlorobenzoyl ) -3- ( 2-hydroxy~4-tr if luorome thyl-
phenyl ) urea r

inhibit tumor growth in several test models.

To determine the inhibitory activity of the com-
pounds in vivo, three test models have been used:

I* B-16 malignant melanoma in C57BI*/6 mice.
To study the effect of drugs or other agents on growth of
tumors in vivo, a metastatic tumor such as B— 16 melanoma
has the advantage of providing both a primary site and
microscopic foci of metastatic tumors,

XI. Ridgeway Osteogenic Sarcoma (KOS) in AKR mice.
The advantage of the use of such a localized, non-meta~*
static tumor for studies on drug effects is that pertuba**
tions of tumor growth and longevity of the mice are attri-
butable to a direct effect on the primary tumor.

Ill* Slcin carcinoma {Ca 1025) in AKR mice.
This tumor represents a second histologic cell type ari-
sing from skin and is less malignant than malignant mela-
noma #

0107214

— 3""

The compounds were tested by intraperitoneal in-
jections of suspensions of the compounds in an aqueous
solution of polyvinyl pyrrol idone. Doses and schedules of
injection were varied, but no optimal dose- schedule has as
yet been determined for either compound.

The lethality of the compounds wth the exception
of PH 60-44 was extremely low. Injection of the compound
was essentially non-lethal even in massive doses of 100
mg/mouse.

Lethality for PH 60-44 was observed at a much
lower dose- Multiple injections for a total dose of 25-30
mg during a five day period resulted in 10-15% mortality*
Therefore, the experiments included injections of 10 mg
each for 3 injections or 5 mg each for 5 injections •

The compounds were tested according to the fol-
lowing procedures;

A* C57BL mice with malignant melanomas (B-16)
Mice with malignant melanomas were injected with 20 mg or
40 mg diflubenzuron in suspension or with the suspension
medium (blank). In two of the experiments an initial in-
jection of 20 mg of diflubenssuron resulted in an 11*7% and
a 13.4% reduction in tumor size within 24 hours of treat-
ment, as compared to a 60.7% and a 34% increase, respecti-
vely, in tumor size for blank-treated controls (Table I).
This effect is also emphasised by a large increase in
doubling time (i.e., mean time in days for the tumors to
double in mass calculated on the basis of the change
within 48 hours after treatment) from 1*9 days for the
blank-treated control to 10,4 days for di f lubenssuron-
-treated animals (Table I). Injection of 40 mg resulted in
a greater effect (initial decrease by 22% and a negative
doubl ing t ime ) .

0107214

-4-

TABLE I*

treatment

% change in

tumor mass

initial doubling
time (in davs)

day 1

day 2

0-2 days

20 mg diflu-
benzuron

-11,7(26)

18.6(26)

10.4(26)

0*2 ml blank

60.7(16)

128.6(16)

1* 9(16)

20 mg diflu-
benznron

-13.4(22)

0.2 ml blank

34*0(9)

40 .mg diflu-
benzuron

-22.4(8)

-14*7(8)

-6.7(8)

0.4 ml blank

31.4(9)

85,1(9)

2*5(9)

Xn a further experiment injections of 20 mg each

on day 0 and day 2 or 4 resulted in a further delay of
tumor growth (Table II). After a single injection the
tumors remained reduced for about 2 days. However, a re-
peat injection of 20 mg on day 2 or 4 resulted in reduced
rate of growth through about the 6th day.

TABIDS II

treatment

% chancre i

c ma & &

day 1

day 2

day 3

day 4

day 5

day 6

day 15

2 x 20 mg.
di f lubenzuron

"*11 . 7
(26)

18.6
(26)

92.6

(16)

143
(26)

178
(10)

264
(26)

1187
(16)

2 x 0.2
blank

60,7
(16)

128
(16)

176
(11)

247
(5)

314
(16)

437
(16)

1402
(14)

Repeated injections for a total of 100 mg given
in 3 or 5 injections within 6 days after the initial in-

* For tables I-XV the number of tumors measured is given
in parenthesis. Negative values indicate a decrease in
tumor size. A negative doubling time shows time for de-
crease to one— half sis:e.

0107214

-5-

jection reduced tumor growth through the 11th day (Table
III), Between days 12 and 19 the tumor growth was ap-
parently but not significantly less than for controls.
Treated tumors, however, remained virtually static after
the 19th day and the increases for davs 20 and 21 were
less than for the blank-treated controls.

TABLE III

treatment

& nhanae in tumor mass

day 1

day 2

day 5 J

day 7

day 11

day 12

100 mg of
dif lubenzu™
ron in 3 or
5 xni .

-15.8
(30)

-14,7
(8)

106
(30)

279
(18)

358
(ID

478
(22)

1.0 ml blank 1
in 3 or 5
in j .

32.7

(IS)

85*1
(9)

202
(18)

444
(9)

562
(9)

647
(9)

day 13

day 15

day 19 *

day 20

I day 21

559
(30)

864
(30)

1308
(29)

1220
(27)

1460
(27)

753
(18)

1026
j (18)

1589
(16)

1860
(16)

2206
(16)

Similar experiments as were done with diflubenzu-
ron were carried out with PH 60-44*

The initial injection of PH 60-44 on day 0 into
mice with malignant melanomas caused stasis in tumor
growth for about 24 hours (Table IV). Second and third
injections, on days one and five, respectively, however,
had little or no effect on subsequent tumor growth (Table
IV) . Tumor growth at 5 days or later was unaltered as evi
denced by percent increase in tumor mass.

TABLE IV

treatment

%

change

in tumor mass

day 1

day 5

day 6

day 7

day 11

day 14

day 21

3 x 10 mg
PH 60-44

-0.8
(11)

182
(11)

240
(11)

317
(11)

885
(11)

1251
(11)

2834
(11)

3 x 0.1

31.8

206

334

40 3

920

1292

2315

(10)

(10)

(10)

(10)

1 do)

(10)

(10)

:

0107214

6"~*

Also similar experiments have been carried out
with the above identified compounds 2), 3) r 5), 6) and 10)
to 19),

Comparisons were made between treated tumors and
control tumors with a similar mean mass-

The compounds 3), 5), 6) lo), 11 ), 12), 15) and
19) were given in doses of 20 mg each on days 0, 1, 2, 3
and 4. Compounds 13), 14), 16), 17) and IS) were given in
doses of only 10 mg each on days 0, L, 2, 3 and 4. Finally
compound 2) was given in doses of 20 mg each on days 0 r 1
and 2*

The results are summarized in table V.

TABLE V

Treatment

Mean tumor

Mean tumor

% Change in

Mass Day 0

Mass Day 1

Tumor Mass

PVP (17)

224

328

* 40

comp * 2 )

[16)

265

191

- 28

comp • 3 )

C17)

137

127

- 4

comp * 5 )

(14)

198

- 8

comp . 6 )

(10)

232

229

*+* 3

comp • 10 )

203

183

- 10

comp • 11 )

(20)

216

193

comp • 12 )

(15)

180

148

» 16

comp * 1 3 )

(30)

193

205

4- 9

comp * 14 )

(37)

343

359

4- 3

comp -15)

(17)

155

151

- 3

comp . 16 )

(29)

232

231

0

comp. 17)

(28)

327

347

4- 9

comp* 18)

(32)

324

314

- 3

comp* 19)

(19)

189

159

- 15

B . Ridfle way O s teogenic Sar coma ( ROS) in AKR mi ce .

Doses of di flubensuron of 20 mg on day 0 (Table
VI )# 20 mg each on days 0 and 3; and 20 mg each on days 0,

0107214

-7-

1, 2, 5 and 6 (Table VII) were used in this test model. As
with the malignant melanoma model, a reduced rate of
growth was observed 24-48 hours after an initial injection
of dif lubenssuron (Table VI) but tumor sisse did not de-
crease*

TABI*E VI

treatment

% chancre

in tumor

mass

day 1

day 2

20 nig dif luben^uron

14.4

(29)

48,5

(19)

0„2 ml blank

43.5

(17)

80. 0

(11)

Repeated injections reduced the rate of tumor
growth, but not beyond the approximate times of treatment
(Table VII),

TABLE VII

treatment

3. Grange

in tumor mass

day 1

day 2

day 3

day 4

day 5 _j

day 7

2 x 20 mg
dif lubenz.

32.0
(10)

104
(10)

213
(10)

298
(10)

511
(10)

2 x 0.2 ml
blank

73.7
(6)

AM*

223
(6)

250
(6)

354
(6)

562
(6)

5 x 20 rog
dif lubenz.

12,2
(10)

43.3

(10)

MM*

285
(10)

423
; (10)

5 x 0,2 ml
blank

(6)

91,4
(6)

479
(6)

697
(6)

i day 14

day 21

1682
(10)

4051
(10)

1847
(6)

3933
(6)

2495
(10)

8880
(8)

2349
1 (6)

1

6552
(6)

I

0107214

-8-

The effects were lees (or the treatment was
not as effective) in this tumor model than in the malig-
nant melanoma model,

A single dose of PH 60-44 resulted in reduced
rate of tumor growth at 24 hours (Table VIII). This table
shows the results obtained in two experiments in which 30
mg total of PH 60-44 was injected into AKR mice with ROS.
However* the schedules of injections were days 0, 1 and 2
for the first one, and days 0 r 3 and 5 for the second one*
Tumors of the first experiment responded very well resul-
ting in tumor stasis or only a slight increase at 1 and 2
days, and reduced percentage increase through 11 days* On
the other hand, the protracted schedule (days 0, 3 and 5)
was not effective beyond the initial injection.

The initial effect of treatment with PH 60-44
for both experiments in this model was slightly greater
than the initial effect of dif lubenzuron in the same model,.

TABLE VI XI

treatment

% chancre in ti

amor mass

day 1

day 2

day 3

day 6

day 7

day 11

3 x 10 mg
PH 60-44

10.8
(13)

2*7
(13)

17.7

(13)

196
(13)

314
(13)

808
(13)

3 x 0.1 ml

74.0
(8)

118
(8)

■15

(8)

435
(8)

568
(8)

1240
(8)

3 x 10 mg
PH 60-44

0.6

(16)

58.4
(16)

217
(16)

293
(16)

767
(16)

3 x 0 • 1 ml
blank

10*7
(6)

61*1
(6)

190
(6)

377
(6)

789
(6)

day 14

day 21

1501
(13)

3524
(13)

1813
(8)

3385
(8)

1277
(16)

3452
(16)

1323
(6)

3586
(6)

0 107214

HK

T

C. Skin carcinoma (Ca 1025) in AKR mice*

A single injection of di f lubenzuron in this
model resulted in a decrease in tumor mass at 24 'hours and
a decreased rate of growth at 48 hours (Table IX) -

TABLE I X

treatment j % change in tumor mass

day 1

day 2

20 mg dif lubenzuron

"3*8

(35)

1 9 * 9

(7)

0*2 ml blank

45-9

(26)

38.2

<8>

However, multiple doses did not alter tumor
growth significantly beyond the initial injection.

The compound PH 60-44 was also tested in this
model* Mice were treated with 5 mg each in suspension or
0*05 ml of medium on days 0, 1, 2 f 3 and 4 or with 10 mg
in suspension or 0*1 ml of medium on days 0, 1 and 5.

An initial injection of 5 or 10 mg resulted in an
8*2% decrease in tumor mass on day 1 and a second injec-
tion on day 1 resulted in a 5*7% decrease by day 2, where-
as blank-treated controls increased by 54*4% on day 1 and
76*6% by day 2 (Table X),

Multiple injections during the first 5 days after
the initial injection resulted in reduced tumor growth
throughout the 21-day testing period*

TABLE X

treatment I

day 1

5 x 5 mg -8.2
or 3 x 10 (17)
mg PH 60-44

5 x 0*5 ml 54*4
or 3 x 0*1 (16)
ml blank

% change in tumor mass

day 2 day 3

-5*7
(11)

76.6
(3)

12.0
(11)

100
(9)

day 4

28*7

(11)

139
(9)

day 7

124
(17)

465
(16)

day 8

186
(17)

547
(16)

0107214

-10

treatment

% change in tumor mass

day 14 day 18

day 21

713
(17)

1357
(17)

1889
(17)

1332
(16)

2116
(15)

3001
(15)

It appears from the results of the above test
models that the tu^oricidal effect of dif lubenzuron is
probably dose-schedule dependent* Increased doses and pro-
tracted doses of diflubenzuron in the malignant melanoma
model enhances and prolongs tumor inhibition.

PH 60-44 has a moderate effect on malignant mela-
noma tumors in that the tumors fail to grow for 24 hours
after a single injection. However, PH 60-44 produced a
strong a prolonged effect in Ca 1025 skin tumors.

To test the possibility that one or more of the
known metabolites of known insectici dally active 1-ben-
zoyl-3-phenyl ureas have an antitumor activity, a series
of experiments were carried out to determine the effects
of metabolic function inhibitors and inducers on the ac-
tion of diflubenssuron in the animal model with malignant
melanoma tumors.

Mice of the C57BL/6 strain were injected with 1 x
10 malignant melanoma tumor cells, and tumors of ap-
proximately 50-500 mg developed in the mice at the sites
of injection in about 14 days. Animals with growing tumors
were treated with 100 mg of di f lubenzuron alone (suspended
in an aqueous solution of polyvinylpyrrolidone) or in com-
bination with a metabolic inhibitor or a metabolic inducer
according to the schedule given in Table XI.

Cobaltous chloride was used as a metabolic inhi-
bitor* This compound inhibits the monooxygenase activity
in liver microsomes.

As metabolic inducers phenobarbi tal and 3-methyl-
cholanthrene were used.

Phenobarbi tal is a general mixed function oxidase
inducer and increases the metabolism of a wide variety of

substances. It causes a large, increase in biphenyl 4-hy-

0107214

-11-

droxylase activity, but only a small increase in 2-hy-
droxylase activity*

3-Methylcholanthrene is an inducer of microsomal
mixed function oxidase activity. O^is is mainly responsi-
ble for hydroxylation (oxidation) of promatic compounds.
3~Methylcholanthrene induces 2 -hi uroxylase activity but
not 4-hydroxylation*

TABLE XI

Treatment Schedule and dose

treatment day-4

100 mg
dif luben™*
zuron

4 mg phe- 1 mg
nobarbit, and

100 mg di-
f lufoenKu-
ron

6.4 mg 3- 1.6mg
mehylcho-
lanthrene and
100 mg di-
£ lubenzu-
ron

2 * 4mg CO*™ - "™ *"*
baltous
chloride and
100 mg di-
f lubenzu-
ron

day*»3

1 mg

1 • 6rag

day- 2

day-1

day 0

20 mg

day 1

20 mg

day 2

20 mg

day 3

20 mg

1 mg

1 mg

20 mg

20 mg

20 mg

20 mg

1 . 6mg

1 * 6mg

20 mg

20 mg

20 mg

20 mg

1 . 2mg

1 • 2mg

20 mg

20 mg

20 mg

20 mg

day 4

20 mg
20 mg
20 mg
20 mg

0107214

-12-

The tumors of the experimental groups were mea-
sured and growth rates were compared.

D. Cobaltous chloride as a metabolic inhibitor.

Treatment with dif lubenzuron alone gave a reduc-
tion in tumor mass on days 1-3. However, when cobaltous
chloride was given before administration of dif lubenztaron
the inhibitory effect of dif lubenzuron Was decreased
(Table XII) .

It can be concluded from this experiment that an
action to reduce monooxygenase activity of liver micro-
somes also reduces the antitumor activity of diflubenzu-
ron* These data support the concept that enzymatic activi-
ty in liver chromosomes results in a metabolic product of
dif lubenzuron that is a more active anti-tumor agent than
the parent compound*

TABLE XXX

Treatment

% change in

tumor

mass

day 1

day 2

i day 3

day 4

day 5

day 10

day 14

100 mg di-
f lubenzuron

*™ rfj* dip * 7

(17)

-8.6
(17)

1 -8.4
(17)

26.3
(17)

63.9
(17)

266
(17)

378
(12)

100 mg di~
f lubenzuron
2.4 rag co-
baltous
chlor ide

14.4
(16)

20.9
(12)

1 36.4
(12)

39*3
(12)

59.7
(12)

306
(12)

782
(9)

jj£ » Metaboli c inducers

1 * Phenobarbital

Xf, indeed, effects of dif lubenzuron are directly
related to enzymatic activity, an enhanced enzymatic acti-
vity obtained by administration of an oxidase inducer
could increase the antitumor activity of dif lubenzuron*

In a first experiment phenobarbital was used to
enhance liver metabolism, and consequently to increase the

0107214

enzymatic activity of liver microsomes* Phenobarbi tal was
injected into tumor bearing mice prior to the standard
treatment with dif lubenzuron.

As shown in Table XIII phenobarbi tal, which
causes a large increase in biphenyl 4*~h>droxylase activi-
ty, but only a small increase in 2 -hydroxy la t ion, did not
enhance the antitumor activity of dif lubenzuron*

TABLE XIII

treatment

% change in tumor mass

day 1

day 2

day 3

day 4

day 5

day 10

day 14

100 mg di-

-22*7

-8.4

26.3

63*9

266

378

flubenzu-

(17)

(17)

(17)

(17)

(17)

(17)

(12)

ron

.4 mg phe-

nobarbi tal

-19.4

-10.4

0.8

21*7

72.1

293

572

100 mg di-

(23)

(23)

(23)

(23)

(23)

(23)

(10)

flubenzuror

a

2. 3-Methylcholanthr ene ( 3-MC )

To determine whether increase of 2™hydroxylation
of dif lubensuron enhances the antitumor activity of diflu-
ben&uron, 3-MC was given to tumor-bearing mice before the
standard treatment with 100 mg dif lubenzuron, 3-MC en-
hances 2 -hydroxylase activity but not 4~hydroxylase acti-
vity* As a further control a small group of tumor-bearing
mice was given 3-MC alone* 3-MC enhanced the antitumor
effects of diflubenauron throughout the five-day measure-
ment period (Table XIV)*

TABLE XIV

treatment

% change in tumor mass

day 1

day 2

day 3

day 4

day 5

100 mg diflu-

-20*2

-12.5

7.9

24.4

54*7

benzuron

(22)

(22)

(22)

(22)

(22)

0107214

—14—

treatment

6,4 mg 3-meth-
ylcholanthrene
and 100 mg
di flubenzuron

6.4 mg 3~*raetli-
ylcholanthrene

% change in tumor mass

day 1

-29.2
(34)

122
(6)

day 2

***23 • 5
(34)

223
(6)

day 3

-20,6
(3? v

364
(6)

day 4

-15,1
(29)

417
(6)

day 5

5*7
(21)

363
(6)

It is also clear rrom the table that 3-MC alone does not
inhibit tumor growth.

F. Administration of 2-hydroxy- and 3-hydroxydif lubenzuron

As a further experiment to verify the results of
the tests described under A and B, groups of tumor-bearing
mice were given 100 mg of dif lubenzuron, 100 mg of the
3-hydroxy metabolite, or 100 mg of the 2-hydroxy metabo-
lite, respectively, 20 mg each on day 0, 1, 2, 3 and 4*

The 2-hydroxy metabolite was more effective, for
the first 48 hours after treatment, than the parent com*
pound dif lubenzuron (Table XV). Moreover, the 3-hydroxy
metabolite was not active at all.

TABLE XV

treatment

*

% change in

tumor mass on

day 1

day 2

day 3

day 4

day 5

day 11

100 mg di-
flubenzuron

(12)

-7.2
(12)

34.6
(12)

92
(12)

1 9 2
(12)

891
(12)

100 mg 3-OH
dif lubenzu-
ron

39.5
(12)

52.4
(12)

(12)

149
(12)

223
(12)

930
(12)

100 mg 2-OH
di f lubenzu-
ron

-23.4
(12)

-17.4

(12)

25.1
(12)

74.7

(10)

127
(10)

699
(10)

These results support the previous data which indicate

that the 2-hydroxy metabolite of dif lubenzuron is more
tuntoricidal than dif lubenzuron itself.

G * Bf feet of some compounds on malignant melanomas (B-16)
in vitro.

0107214

*** wIL

Cells of malignant melanomas B-16 grow as a mono-
layer. The doubling time of this type of cells in a cell-
-culture is 12 to 16 hours. For this experiment 6 multi-
well tissue culture plates having a flat bottom with a

2

surface area of 6 cm appeared to be suitable and have
been used*

On day 0 a quantity of melanoma B-16 cells were
brought on the cell-culture plates*

The test compounds were sonicated and then incu-
bated without cells for 3 hours at 37 tf c.

Then the compound to be tested was administered
to the plates with melanoma B-16 ceils in the desired
quantity (each test was done in triplicate). Thereafter
the plates were incubated in a GO^-incubator for 20
hours at 37 & C. Then the incubation was stopped by removing
the culture medium containing the test compound, the cells
were washed twice/ and fresh culture medium was added*

48 Hours after starting the incubation step the
number of cells on each plate was measured by means of a
microoell Coulter Counter- The results so obtained have
been expressed as "percentage of controll cell growth" *
The results are summarized in Table XVI.

TABiLE XVI

Comp . No

Percentage of controll cell growth when
tested in an amount of

3
5
8
9
20
21

5 x 10^ --tag/ ml

78
33
98
51

100
91

101

5 x 10 3 ✓ug/ml

41

a

86
53
70

71

in the above experiment the compounds to be tes-
ted have been used in the form of a suspension* Since it
is not quite clear how much of the active compounds is

dissolved^ the above results cannot be compared very well

0107214

-16-

Therefore a number of compounds lias been dis-
solved in ethanol. The results so obtained in the same
test as described above, i.e. against melanoma B-16 cells
are summarized in Table XVII.

TABIxE XVII

Comp • No

Amount ot act. comp.
(ug/ml)

Percentage of controil cell
growth

(solution)

(suspension)

1

50

48

79

2

200

72

6

300

95

33

50

39

103

7

300

0

103

8

300

0

63

21

300

43

50

Ethanol itself has no effect on the growth of
melanoma B-16 cells.

0107214

17-

WHAT IS CLAIMBD IS:

1 * A composition having antitumor activity cha-
racterized in that it contains at least one known insecti-
cidally active l-benzoyl-3-phenyl urea or a known metabo-
lite thereof, having antitumor activity, as active compo-
nent, in association with a pharmaceutical^ acceptable
carr xer *

2* A composition according to Claim 1, characte-
rized in that it contains at least one of the compounds

1) l-{2, 6-di£ luorobenzoyl ) -3~ ( 4-chlorophenyl )urea;

2 ) 1- ( 2 , 6-di f luorobenzoyl ) -3- C 3 # 5-dichloro-4- ( 3-chloro-5-
-trifluorome thy 1 - 2 -pyr i dyl oxy ) phe nyl ] ur ea ;

3 ) 1~ ( 2 , 6-di £ luorobenzoyl ) -3-[ 3-chloro~4- { 4-chlorophenoxy )
phe nyl 2 ur ea ?

4 ) 1- ( 2 , 6-di fluorobenzoyl ) -3- ( 4-tr i £ luorome thylphenyl } urea ;
5 } 1- ( 2-chlorobenzoyl ) -3- { 4-chlorophenyl ) urea ;

6 ) 1- ( 2-chlorobenzoyl ) -3- { 4-tr i £luoromethoxyphenyl ) urea ;

7 ) 1- ( 2 , 6-di fluorobenzoyl ) -3- { 4-chloro-2-hydroxyphenyl
urea;

8 ) 1- ( 2 , 6-di f luorobenzoyl ) -3- ( 4~chloro-3-hydroxyphenyl ) urea

9 ) 1- ( 2-chlorobenzoyl ) -3- ( 2-hydroxy-4~tr i f luoromethoxy-
phenyl)urea.

10 } 1- ( 2 , 6-di f luorobenzoyl ) -3- { 4-cyanophenyl ) urea ;

11 ) 1- { 2 , 6-di f luorobenzoyl ) -3- [4- (1,1,2,3,3, 3-hexaf luoropro-
poxy ) phe nyl ] ur e a ;

12 ) 1- ( 2 , 6-di £ luorobenzoyl ) -3™ ( 4-hydroxyphenyl ) urea ?

13 ) 1- ( 2 , 6-di f luorobenzoyl ) -3- (4-cyclohexylphenyl ) urea ;

14) 1- ( 2 , 6-di f luorobenzoyl ) -3- ( 4-tr i fluoromethoxyphenyl ) urea

15 ) 1- { 2 , 6-di f luorobenzoyl ) -3- ( 3 , 4-dichlorophenyl ) urea ;

16 ) 1- { 2-chlorobenzoyl ) -3- ( 4-tr i f luoromethylphenyl ) urea ;

17 ) 1- (2, 6-dichlorobenzoyl ) -3- { 4-chlorophenyl ) urea ;

18) 1- ( 2 , 6-dichlorobenzoyl ) -3- (4-tr i fluorome thylphenyl ) urea

19 ) l-benzoyl-3- (4-chlorophenyl ) urea ;

20 ) 1- ( 2 , 6-di fluorobenzoyl ) -3- { 2-hydroxy-4-t r i fluorome thyl-
phenyl) urea;

21 ) 1- ( 2-chlorobenzoyl ) -3™ ( 2™hydroxy-4-tri f luorome thyl-
phenyl) urea;

0107214

3* A method of combating tumors in mammals, cha-
racteri&ed in that a therapeutically effective quantity of
a known insecticidally active 1 -benzoyl- 3~pheriyl urea or a
known metabolite thereof is administered in association
with a pharmaceutically acceptable carrier*

4, A method according to Claim 3, characterized
in that a therapeutically effective quantity of

1 ) 1- ( 2 , 6-di fluor obenzoyl ) -3*- { 4*~chlorophenyl ) urea ;

2 ) 1» { 2 , 6-di f luorobenzoyl ) -3-E3 , 5-dichloro-"4- ( 3~chloro-5~
~tr i f luorome thyl-2-pyr idyloxy ) phenyl 3 urea ;

3 } 1~*( 2 , 6**di£ luorobenzoyl )~3-[3-chloro-4-(4-chlorophenoxy)
phenyl 1 urea ;

4 ) 1- ( 2 , 6-di fluor obenzoyl ) "-3™ (4-tr i f luorome thylphenyl ) urea ;

5 ) 1— ( 2— chlorobenzoyl ) -3- ( 4-chlorophenyl } urea ;

6 ) 1- ( 2*-chlorobenzoyl ) -3- { 4-tr i f luorome thoxyphenyl } urea;

7 ) 1- ( 2 , 6-di f luorobenaoyl ) -3- { 4-chloro-2~hydroxyphenyl urea

8 ) l-( 2 , 6~di f luorobenzoyl ) -3™ { 4-chloro-3-hydroxyphenyl ) urea

9 ) 1~( 2-chlorobens5oyl)-3-( 2~hydroxy~4-tri£ luorome thoxy-
phenyl) urea

10 ) 1- { 2 , 6-dif luorobenaoyl } -3- ( 4-cyanophenyl ) urea ?

11 ) 1™ ( 2 , 6-di fluorobenzoyl ) -3-H4- (1*1,2,3,3, 3™hexaf luoropro-
poxy ) phenyl ] ur ea ;

12 ) 1- C 2 , 6-di f luorobenzoyl } -3- ( 4Hbtydroxyphenyl ) urea;

13 ) 1- ( 2 , 6-di f luorobenaoyl ) -3- (4™cyclohexylphenyl ) urea;

14 ) 1- ( 2 , 6-di £ luorobenzoyl } -3- ( 4-tr i f luorome thoxyphenyl ) urea

15 ) 1- ( 2 , 6-di f luorobenaoyl ) -3- ( 3 , 4-dichlorophenyl ) urea ;
16 ) 1*- ( 2~chlorobenzoyl ) -3™ ( 4-tr i f luorome thylphenyl ) urea ?

17 ) 1- { 2 , 6-dichlorobenzoyl ) -3- { 4-chlorophenyl ) urea ;

18 ) 1- { 2, e-dichlorobenzoyl ) -3- ( 4^tr if luorome thylphenyl ) urea
19 } 1 ^benzoyl *3 ( 4-chlorophenyl ) urea ;

20 } 1- ( 2 , 6-di f luorobenaoyl } -3- { 2-hydroxy-4-tr i f luoromethyl-
pheny 1 ) urea ;

21 ) 1- ( 2-chloroben5s;oyl ) -3 - { 2-hydroxy-4-tr i f luor ome thyl-
phenyl ) urea;
is used.

5. 1 -Benzoyl— 3 -phenyl urea compounds having anti-
tumor activity.

010721*

6. Compounds according to Claim 5 being

1) l-~(2, 6-~di£ lviorobenzoyl } - { 4*chlorophenyl ) urea ?

2 ) 1- ( 2 , 6-di f luor obenzoyl } -3- [ 3 , ichloro-4~ ( 3-chloro-5-
-trif luor ome thy 1 ~2™pyridyloxy) phenyl 3urea ;

3 ) 1- ( 2 , 6-di f luorobenzoyl ) -3-L 3-chloro { 4-chlorophenoxy )
phenyl] urea;

4 ) 1- ( 2 , 6-di f luorobenzoyl ) -3- { 4-tr i f luoromethylphenyl ) ur ea ;

5 ) 1- { 2-chlorobensoyl ) -3- ( 4-chlorophenyl } urea ;

6 ) 1- { 2-chlorobenzoyl ) -3- ( 4-tr i f luoronte thoxyphenyl ) urea j

7 ) 1™ ( 2 , 6™di f luorobexxaoyl } -3- ( 4-chloro-2--hydroxyphenyl urea

8 ) 1- ( 2 , 6-dif luoroben&oyl ) -3- ( 4~chloro~3--hydroxyphenyl ) urea

9 ) 1™ ( 2-~chlorobenj&oyl ) -3- ( 2-hydroxy~4~-t r i fluoroiue thoxy
phenyl ) urea .

10} l-{2, 6~dif luoroben£oyl)~3-(4-cyanophenyl }urea;

11 ) 1- £ 2 , 6-d i fluor obenzoyl ) -3* [4~(X,1#2,3,3, 3-hexaf luoropro-
po xy ) phe ny 1 3 ur ea ?

12) 1»(2, 6-dif luorobenaoyl ) -3- (4-hydroxyphenyl)urear

13) 1** ( 2, 6~di£ luorobenjsoyl )-3- (4-cyclohexylphenyl )urea ;

14} 1- ( 2 , 6~di f luorobensoyl ) -3™ ( 4-tr i f luor orae thoxyphenyl ) urea

15 ) 1- ( 2 , 6-d i f luorobenzoyl ) ™3~ { 3 , 4™dichlorophenyl ) urea ;

16 ) 1- ( 2-chlorobenzoyl ) -3~ £ 4- 1 r i f luor ome thy 1 phenyl ) urea ;

17 ) 1- { 2 , 6-d ichlor obenzoyl ) -3- ( 4-chlorophenyl ) urea ;

IB) 1™ C 2 , 6-di chlor obenzoyl ) *-3- ( 4-tr i f luoromethylphenyl ) urea
19 5 l-*ben£oyl~3 - ( 4—chlorophenyl ) urea ;

20 ) 1- ( 2 , 6-di f luorobenzoyl ) -3- < 2-hydroxy-4-tr i f luor ome thy 1-
phenyl )urea;

21 ) 1- ( 2~chlorobenzoyl )-3-{ 2-hydroxy-4-tri£luoromethyl-
phenyl)urea;